Molecules that absorb and emit light in the near-infrared (NIR) wavelengths have become the central focus of numerous analytical, biological, and spectroscopic studies because their spectral, chemical, and biological properties facilitate the detection of minute molecular processes in solutions and living tissues. Using NIR fluorescent technology allows almost the complete elimination of background from auto fluorescence of biomolecules. Also the background from the scattered light from the excitation source is greatly reduced since the scattering intensity is proportional to the inverse fourth power of the wavelength.
Cyanine dyes have been widely used for labeling biomolecules including antibodies, DNA probes, avidin, streptavidin, lipids, biochemical analogs, peptides, and drugs, as well as for a variety of applications including DNA sequencing, DNA microarray, western blotting, flow cytometry analysis, and protein microarrays to name a few. The excellent safety profile of the NIR heptamethine cyanine fluorochrome indocyanine green (ICG) in humans has spurred interest in the development of ICG derivatives, including Cy dyes and cypate for in vivo molecular imaging by NIR optical methods. But the low fluorescence quantum yield, short fluorescence lifetime, propensity to photobleach, and poor chemical stability of these dyes have limited their use in chemical and life sciences.
Attempts to overcome these limitations have led to monofunctional cyanine dyes produced using highly toxic organic solvents resulting in a poor yield and cyanine dye stability concerns. Further, currently available NIR dyes possess a conformationally constrained central meso-chloro cyclohexenyl group, which is functionalized for labeling or available for reaction with diverse molecules by substituting the chloro with heteroatoms such as O, S, and N. These heteroatoms introduce a high degree of chemical and biological instability. The art is in need of stable cyanine dyes that can be produced cost-effectively without the use of toxic solvents.